(a) Field of the Invention
The invention relates to a method for the in vitro aseptic mass production of endomycorrhizal fungal propagules, mainly spores, using a transformed root or a root-hypocotyl explant cultivated in a two-compartment Petri dish or bioreactor.
(b) Description of Prior Art
Arbuscular mycorrhizal fungi (AM) are beneficial fungi in that they colonize the cells of feeding roots of plants and stimulate uptake of phosphorus from the soil. Hyphae of the fungus grow outwardly from the root, well beyond the phosphate depletion zone (the zone from which the available phosphate has already been consumed by the plant). Selected AM fungi have been shown to enhance the growth of numerous plants of economic importance (Bethlenfalvay, G. J., 1992, Amer. Soc. of Agr., Special Publication #54, Madison Wis., pp. 1-27), including agronomic, horticultural and forest plant.
Furthermore, it has been observed that colonization by an endomycorrhizal fungus may protect the roots of the mycorrhized plant from pathogens in the soil (Linderman, R. G., 1992, Amer. Soc. of Agr., Special Publication #54, Madison Wis. pp. 45-70).
However, despite the large amount of work carried out to date, the main difficulty in applying endomycorrhizae is that, it is not known how to produce their propagules in large quantities for commercial application (Wood, T. and B. Cummins, 1992, Mycorrhizae Functioning; an Integrative Plant-Fungal Process, pp. 468-487, Ed. M. F. Allen, Academic Press, New York). It is believed that no one has ever succeeded in cultivating them in a sterile medium without the host plant (Becard, G. & Y. Piche, 1992, Methods in Microbiology, Vol. 24, 90-108, Ed. Norris, Read & Varma, Academic Press, London).
Unlike most symbiotic micro-organisms which are parasitic or saprophytic, AM fungi are obligate biotrophs which have so far resisted all attempts to be cultivated axenically (in pure culture); (Williams, G., 1992, Methods in Microbiology, Vol. 24, 203-220. Ed. Norris, Read & Varma, Academic Press, London). This lack of independent growth has not prevented vesicular-arbuscular mycorrhizal fungi from becoming distributed world-wide as a symbiotic partner of most vascular plants, under a wide variety of pedologic and climatic conditions (Bethlenfalvay, G. J., 1992, Amer. Soc. of Agr., Special Publication #54, Madison Wis., pp. 1-29).
The large number of attempts at mycorrhization which have been carried out so far have consisted of using inoculums prepared from complete plants which are cultivated in pots or a greenhouse. Inoculation is nearly always carried out with specially gathered mycorrhizal roots, or sometimes with a suspension of spores, Jackson et al. (Jackson et al, 1972, Soil Sci. Soc. Amer. Proc., Vol. 36, 64-67) used lyophilized roots, while Hall (Hall I. R., 1979, Soil Biol. Biochem., Vol. 11, 85-86), recommends using soil pellets mixed with infected roots.
In all cases, one of the problems encountered is the possibility of introducing contaminants into the soil through inoculation of the endomycorrhizal fungi, particularly when for reasons of plant health it is necessary to disinfect the soil before planting, thereby resulting in destruction of the endomycorrhizal fungi naturally present. Moreover, the time required to produce an inoculum is generally two-four months, which is long enough to produce many contaminants. Such problems have hindered commercialization of the inoculums. In addition, the inoculums cost at least as much as, or more than, the fertilizers which would be required to obtain the same yield. Consequently the large scale use of endomycorrhizal fungi has not yet been adopted in agricultural practice due to the problems of producing an inoculum in large amounts, free of contaminants, yet easy to store and easy to handle on the land (Wood, T. and B. Cummins, 1992, Mycorrhizae Functioning: an Integrative Plant-Fungal Process, pp. 468-487, Ed. M. F. Allen, Academic Press, New York).
Cultivation of vesicular-arbuscular mycorrhizal fungi under axenic conditions (i.e. without a host plant) continues to be a preoccupation and represents one of the most challenging goals of modern plant biology. Using roots growing in vitro as plant partners, Mosse provided new scientific insights on this question when they succeeded in establishing arbuscular mycorrhizal symbiosis in vitro (1975, Physiol. Plant Pathology, Vol. 5, 215-223). However, colonization and sporulation were limited in these monoxenic systems. The VAM have also been established in solution culture (Howeler et al, 1982, New Phytol., Vol. 90, 229-238; Mosse et al, 1984, Can. J. Bot., Vol. 62, 1523-1530).
For example, Mosse reported approximately 50% colonization of Zea mays roots after nine weeks in a nutrient film system. The inoculum produced in their nutrient system, however, was primarily colonized root material. In general, sporulation in solution culture systems has been poor. This is due to the fact that VA-mycorrhiza are inhibited by excessive moisture and poor aeration in the natural environment.
In recent years, culture of isolated roots was given a new impetus with the use of roots genetically transformed by the Ri plasmid of Agrobacterium rhizogenes (Tepfer, 1984, Cell, Vol. 37, 959-967). Rapid growth of axenic "hairy roots" led Mugnier and Mosse (1987, Phytopathology, Vol. 77, 1045-1050) to inoculate such roots with vesicular-arbuscular mycorrhizal fungi. They observed some vesicular-arbuscular mycorrhizal colonization on transformed roots of Convolvulus sepium inoculated with germinated spores of Glomus mosseae. Some evidence of limited independent growth of G. Intraradices was also observed (Mosse, 1988, Can. J. Bot., Vol. 66, 2533-2540). These authors thus demonstrated that root organ cultures have potential for growing vesicular-arbuscular mycorrhizal fungi in vitro. The method used, however, did not result in the reproducible generation of the entire life-cycle of the fungus including the production of viable spores nor the ongoing cultivation of the symbiotic root-organ system.
Further, Mugnier and Mosse (1987, Phytopathology, Vol. 77, 1045-1050) do not teach a method for the co-culture of the symbiotic combination of the root and the endomycorrhizal fungi and wherein the endomycorrhizal fungi has the opportunity to grow into a root-free compartment to produce the endomycorrhizal spores in mass quantity.
Mugnier et al., in U.S. Pat. No. 4,599,312 issued on Jul. 8, 1986 in the name of Rhone-Poulenc Agrochimie, discloses a method of obtaining endomycorrhiza formation with vesicles and arbuscules in vitro. The method comprises producing dicotyledone roots which have been genetically transformed by inserting genes of root inducing or tumor inducing plasmids into the genome of dicotyledone roots, and then inoculating the cultivated converted roots with pregerminated endomycorrhizal spores. Mugnier et al. discloses the preparation of these converted roots inoculated with endomycorrhizal spores divided in three steps: 1) conversion of the root; 2) cultivation of the converted root; and 3) spore germination and mycorrhization.
They do not disclose the in vitro sporulation of the endomycorrhizal fungi nor the ongoing cultivation of the symbiotic root-organ system. Moreover, no provision is made to create a root-free and root exudate-free compartment for promoting massive sporulation.
Sylvia et al., in U.S. Pat. No. 5,096,481 issued on Mar. 17, 1992 in the name of University of Florida, discloses a vesicular-arbuscular mycorrhizal inoculum composition comprising a host plant roots colonized by at least one species of vesicular-arbuscular mycorrhizal fungus. The colonized roots having a particle size in the range of 33 .mu.m to about 425 .mu.m and a propagule density of about 10,000 to about 1,000,000 vesicular-arbuscular mycorrhizal fungi propagules per gram dry mass of host plant root comprise an effective plant growth enhancing agent which is advantageously and efficiently stored, transported and applied in agricultural methods. It has been discovered that subjecting the colonized roots to methods of size reduction such as a high shear size reduction process in liquid, causes the reduction of the size of the roots while actually increasing the fungi propagule density thereof. This method is not aseptic and allows for contamination to occur.
Wood et al., in European Patent Application No. EP-A-209,627 published on Jan. 28, 1987, teaches a process for the production of mycorrhizal fungi in association with plant root organ culture comprising establishing root cultures and inoculating the established root culture with endomycorrhizal spores. In this method no provision is made to built a barrier preventing root exudates to reach for the extramatrical phase; without such a barrier the numbers of spores produced in the vicinity of the root remains very small and not industrially feasible. However, the amount of spores obtained in the best conditions is in the order of 83.+-.32 spores for 30 ml of medium, which is a minute amount, and Wood et al. mention (page 17, line 24) that 1.7 spores/ml medium are obtained. It would thus be highly desirable to be provided with a method which would allow for the production of 6000-7000 spores/ml medium which are aseptic and readily recovered.
Okii et al., in U.S. Pat. No. 4,945,059 issued on Jul. 31, 1990, disclose a method of producing endomycorrhizal fungal spores using potato roots and a porous amphoteric ion exchanger. However, this process is not aseptic and only allows for minute quantities of spores produced. Here again, no provision is made to create the barrier to root diffusates of the present invention.
There is not disclosed a method for the aseptic in vitro mass production of endomycorrhizal fungal spores.
It would be highly desirable to be provided with a method for the in vitro mass production of arbuscular endomycorrhizal fungal propagules, mainly spores, which would be, contamination-free, simple, inexpensive and effective.